Hindawi

Journal of Immunology Research

Volume 2018, Article ID 2978419, 7 pages
https://doi.org/10.1155/2018/2978419

Research Article
Comparison of RPR and ELISA with TPHA for the
Diagnosis of Syphilis: Implication for Updating Syphilis
Point-of-Care Tests in Ethiopia

Markos Negash , Tadelo Wondmagegn, and Demeke Geremew

Department of Immunology and Molecular Biology, School of Biomedical and Laboratory Sciences, University of Gondar,
Gondar, Ethiopia

Correspondence should be addressed to Markos Negash; markosnegash@yahoo.com

Received 2 February 2018; Revised 24 April 2018; Accepted 27 May 2018; Published 8 July 2018

Academic Editor: David Smajs

Copyright © 2018 Markos Negash et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Syphilis is a sexually transmitted disease (STD) caused by the spirochete Treponema pallidum, and it persists to be a
major public health problem in Africa, including Ethiopia. Syphilis diagnosis is made by either nontreponemal or treponemal
approaches, though in developing countries the diagnosis relies mostly on nonspecific tests due to several reasons. Thus, the
objective of this study was to assess the sensitivity, specificity, predictive values, and agreement of rapid plasma reagin (RPR)
and enzyme-linked immunosorbent assay (ELISA) with Treponema pallidum hemagglutination assay (TPHA) as a gold
standard for the diagnosis of syphilis. Results. The sensitivity, specificity, and positive and negative predictive values of
ECOTEST-RPR were 100%, 80.8%, 76.2%, and 100%, respectively. However, the sensitivity, specificity, and positive and negative
predictive values of DIALAB-ELISA were 98.4%, 94.9%, 92.3%, and 98.9%, respectively. The agreement between DIALAB-
ELISA and Randox-TPHA was excellent (kappa value: 0.96) as compared to ECOTEST-RPR and Randox-TPHA assay (kappa
value: 0.88). Conclusion. We found a characteristically variable performance of DIALAB-ELISA test and the currently available
traditional ECOTEST-RPR test in the study area. The use of ECOTEST-RPR as a diagnostic test is confronted by its false
positivity. Thus, neither the ECOTEST-RPR nor the DIALAB-ELISA test stands on its own to be used either as screening or
confirmatory test for syphilis diagnosis. Consequently, thorough studies should be conducted aiming on a change of the current
diagnostic scheme in the community.

1. Background and observed from the lesion under dark-field microscope

Syphilis is a sexually transmitted disease (STD) caused by
the spirochete Treponema pallidum (T. pallidum) which
can be spread by sexual contact, by blood transfusion,
and via vertical transmission [1]. Syphilis affects 12 million
people annually and results in significant morbidity if not
mortality. In sub-Saharan Africa, syphilis remains a serious
public health problem. Prevalence of active syphilis infec-
tion among African countries showed 12.8% in Tanzania
and Kenya [2, 3]. But the magnitude of syphilis among
blood donors in Gondar (Ethiopia) was 1.3% in 2010 [4].
Syphilis diagnosis can be made by several approaches. In
addition to the mainstay serological diagnostic method,
dark-field microscopy by which the spirochete is examined
is also another method [5–7]. T. pallidum, the etiological
agent of syphilis, produces at least two types of antibodies
in human infections: treponemal antibodies that can be
detected by fluorescent treponemal antibody absorption
(FTA-ABS) and nontreponemal antibody (reagin) that
can be detected by RPR antigen card or VDRL test. Non-
treponemal tests such as the venereal disease research lab-
oratory (VDRL) and rapid plasma reagin (RPR) are based
on the reaction of cardiolipin with nonspecific antibodies
produced in response to syphilitic infection [8]. However,
this test lacks sensitivity and specificity due to several rea-
sons including pregnancy, autoimmune disorders, infec-
tions, and stages of syphilis infection [9, 10]. Therefore,
treponemal-specific tests like enzyme immunoassay (EIA),
2 Journal of Immunology Research
T. pallidum hemagglutination assay (TPHA), microhemag-
glutination, fluorescent treponemal antibody absorption test
(FTA-abs), and the enzyme-linked immunosorbent assay
(ELISA) that detect IgG antibodies to antigenic components
of T. pallidum are used primarily to confirm the diagnosis
of syphilis in patients with a reactive nontreponemal test
[11, 12]. Despite the availability of relatively sensitive tests
and affordable treatment, the disease remains a global health
problem [13, 14].
Syphilis remains a major cause of reproductive morbidity
and poor pregnancy outcomes in developing countries
including Ethiopia. In 80% of infected pregnant women, it
results in stillbirth and spontaneous abortion (40%), perina-
tal death (20%), and serious neonatal infections and low-
birth weight babies (20%) [15–17]. Syphilis has also acquired
a new potential for morbidity and mortality through an asso-
ciation with increased risk for HIV infection [18].
Screening of pregnant women, blood donors, and social
workers (drivers and newly employed social workers) for
syphilis is a routine activity at all healthcare institutions in
Ethiopia. For this purpose, due to cost effectiveness, RPR
which has questionable test performance is usually used as
a screening tool in Ethiopia. However, despite reports of
diagnostic performance provided by the manufacturers, data
on test performance of RPR in the study area remain limited.
Thus, the objective of this study was to assess the sensitivity,
specificity, predictive values, and agreement of ECOTEST-
RPR and DIALAB-ELISA with Randox-TPHA assay as a gold
standard for syphilis diagnosis among syphilis-suspected
patients attending the University of Gondar Hospital
(UGH), Northwest Ethiopia.
2. Methods
2.1. Study Design, Period, and Area. Facility-based cross-
sectional study was conducted in UGH, from November
2015 to June 2016. The University of Gondar Hospital is
one of the pioneer teaching hospitals in Ethiopia, located in
Amhara region, Northwest Ethiopia. The hospital has eight
different laboratory sections, including Serology, which
provides teaching, diagnostic, and research services for the
university community, Gondar town inhabitants, and the
nearby Woreda populations.
2.2. Study Participants. Following acquisition of informed
consent, a total of 160 participants were included in this
study. Out of them, 80 participants were diagnosed as posi-
tive for syphilis infection by the routinely used technique in
the study area (ECOTEST-RPR) at the University of Gondar
Teaching hospital Laboratory. Charts of all patients were
reviewed to assess if specific therapy was commenced. Like-
wise, 80 apparently healthy individuals that do not have
any history of sexually transmitted diseases were recruited
from Gondar Blood Bank Center.
2.3. Specimen Collection and Processing. Blood sample was
collected from each participant and centrifuged until the
serum was separated, and sera were stored at −20°C until
the actual laboratory tests were performed. Before running
RPR, ELISA, and TPHA, the stored serum samples were
thawed at 37°C in a water bath until the ice formed becomes
completely dissolved. Thereafter, RPR, ELISA, and TPHA
were done and results were achieved according to the kit’s
manufacturer’s instructions. A specimen that shows an
equivocal result for any of the tests was retested. This study
was performed according to the Standards for Reporting
Diagnostic Accuracy Studies (STARD) essential items for
reporting diagnostic accuracy studies (http://www.equator-
network.org/reporting-guidelines/stard).
2.3.1. ECOTEST-RPR. [Principle] The antigen used in the
ECOTEST-RPR (Assure Tech, Hangzhou, China) kit is a
modification of VDRL antigen, which contains microparticu-
late charcoal to enhance the visual difference between a pos-
itive and negative result. Patient sera mixed with a fine
particle cardiolipin antigen which has been enhanced with
cholesterol, lecithin, and charcoal will result in a macroscop-
ically visible flocculation-type precipitation if the patient’s
serum contains reagin—an antibody formed against cardioli-
pin (The detailed procedure can be accessed from the manu-
facturer’s instruction, Assure Tech, Hangzhou, China.)
Interpretation for each test was done using controls (positive
and negative) according to the manufacturer’s instruction as
reactive (R), if clumping is seen, or nonreactive (NR)—smooth
suspension, no clumping, or slight roughness.
2.3.2. The DIALAB Syphilis IgG/IgM ELISA. [Principle]
Using the antigen sandwich enzyme-linked method (ELISA),
this syphilis IgG/IgM ELISA test (DIALAB, Germany) can
detect anti-TP antibodies. Polystyrene microwell strips are
precoated with recombinant Treponema pallidum antigens
produced in E. coli. Recombinant TP antigens that are conju-
gated to horseradish peroxidase (HRP) conjugate are incu-
bated in the microwells with the sample. The precoated
antigens indicate the same epitopes as the HRP-conjugate
antigens, but the hosts are different. If anti-TP is present in
the sample during incubation, the conjugated and precoated
antigens will be bound to the two-variable antibody domains,
and what is captured on the solid phase is the specific
antibody-antigen immunocomplex. It is important that chro-
mogen solutions containing tetramethylbenzidine (TMB)
and urea peroxide are added into the wells after the washing
phase to remove sample and unbound conjugates. The color-
less chromogens are hydrolyzed by the bound HRP conjugate
to a blue-colored product when the antigen-antibody-
antigen sandwich complex is present. At this point, the blue
color turns yellow. This occurs after the reaction with sulfuric
acid is stopped. What can be measured proportionally at this
juncture is the amount of antibody in the sample with the
amount of color. Colorless wells indicate negative anti-TP
samples (The detailed procedure can be accessed from the
manufacturer’s instruction, DIALAB, Germany.)
(1) Results Interpretation. Each microplate should be
considered separately when calculating and interpreting
results of the assay, regardless of the number of plates con-
currently processed. The results are calculated by relating
each sample’s optical density (OD) value to the cut-off value
(CO) of the plate. If the cut-off reading is based on a single-
Journal of Immunology Research
filter plate reader, the results should be calculated by sub-
tracting the blank well OD value from the print report values
of samples and controls. In case the reading is based on a
dual-filter plate reader, do not subtract the blank well OD
from the print report values of samples and controls.
2.3.3. Randox TPHA. [Principle] TPHA (Randox Laborato-
ries, UK) reagents are used to detect human serum antibody
to T. pallidum by means of an indirect hemagglutination
(IHA) method. Preserved avian erythrocytes are coated with
antigenic components of pathogenic T. pallidum (Nichols
strain). These test cells agglutinate in the presence of specific
antibodies to T. pallidum and show characteristic patterns in
microtitration plates. Any nonspecific reactions occurring
are detected using the control cells, which are avian erythro-
cytes, not coated with T. pallidum antigens. Nonspecific reac-
tions may also be absorbed out using these control cells.
Antibodies to nonpathogenic treponemes are absorbed by
an extract of Reiter’s treponemes, included in the cell suspen-
sion. Test results are obtained in 45–60 minutes, and the cell
agglutination patterns are both easily read and are long-
lasting. (The detailed procedure can be accessed from the
manufacturer’s instruction, Randox Laboratories, UK.)
(1) Interpretation of Results. When the test well is posi-
tive, the control well should be observed. The control cells
should settle to a compact button. They should not be used
as a comparison for nonreactive serum patterns since the
control cells will give a more compact pattern than the test
cells will. Agglutination in the control well indicates the pres-
ence of nonspecific agglutinins in the sample; the test should
be reported as invalid. A serum that gives this result may be
absorbed using the control cells as detailed under nonspecific
absorption. A doubtful reaction with test cells should be
reported as indeterminate.
2.4. Statistical Analysis. The data was cleaned and double
entered on an Excel Spreadsheet and transported to SPSS
version 20. JavaStat two-way contingency table analysis soft-
ware (http://statpages.org/ctab2x2.html) was also used to cal-
culate sensitivity, specificity, and predictive and kappa values.
The test findings of ECOTEST-RPR and DIALAB-ELISA
were compared with results of the reference method (Ran-
dox-TPHA). The kappa value was determined to evaluate
the agreement between ECOTEST-RPR, DIALAB-ELISA,
and Randox-TPHA.
3. Results
A total of 160 participants were involved in this study. 80
(50%) of them were diagnosed with syphilis using RPR as a
diagnostic test in the study area. However, 80 (50%) of them
were apparently healthy participants and were negative for
syphilis by all types of tests (ECOTEST-RPR, DIALAB-
ELISA, and Randox-TPHA). The participants’ age range
was from 20 to 52 years old, and most of them (77%) were
between 22 to 32 years. Among the participants, 84 (52.5%)
of them were males and 76 (47.5%) were females. Most of
the study subjects (107, 66.9%) were from rural areas of the
nearby inhabitants, and 53 (33.1%) of the subjects were
3
urban residents (Table 1). Among 40 patients who were diag-
nosed as having syphilis by the RPR test, 2 patients had pri-
mary syphilis, and 9 patients had secondary syphilis with
clinical presentation of nonitchy maculopapular rash, condy-
loma lata, and generalized lymphadenopathy whereas clinical
data of the rest RPR-positive patients were not fully docu-
mented on the medical charts.
The sensitivity, specificity, and predictive values of
ECOTEST-RPR and DIALAB-ELISA in this study were eval-
uated by using Randox-TPHA as a gold standard for the
diagnosis of syphilis. Thus, the sensitivity and specificity of
ECOTEST-RPR for syphilis detection were 100 and 80.8%,
respectively. The positive predictive value (PPV) and nega-
tive predictive value (NPV) were 76.2 and 100%, respectively.
The agreement between Randox-TPHA and ECOTEST-RPR
tests was good with a kappa value of 0.88 (Table 2).
Moreover, the sensitivity and specificity of DIALAB-
ELISA for syphilis detection were 98.4 and 94.9%, respectively.
The positive predictive value (PPV) and negative predictive
value (NPV) were 92.3 and 98.9%, respectively. The agree-
ment between TPHA and ELISA tests was nearly perfect with
a kappa value of 0.96 (Table 3).
In this study, we have revised the medical charts of each
participant and found that all ECOTEST-RPR-positive
patients were commenced appropriate medication. More-
over, we found two samples with equivocal test results
for DIALAB-ELISA but reanalysis of these samples by
DIALAB-ELISA and Randox-TPHA provides a positive
result under both tests. Similarly, we reported 15 discrepant
results (i.e., ECOTEST-RPR-positive but DIALAB-ELISA-
negative) as a negative result following reanalysis and verified
as negative by Randox-TPHA.
4. Discussion
Syphilis infection can be diagnosed using either the trepone-
mal or the nontreponemal approach. Nucleic acid amplifica-
tion techniques (NAAT) like polymerase chain reaction
(PCR) have opened the way for the development of highly
sensitive and specific point-of-care tests. Nevertheless, the
use of NAAT methods in developing countries is limited
due to their affordability and cost and the complexity of the
techniques to be used by the existing human resources in
diagnostic areas. Additionally, a definitive syphilitic diagnos-
tic test based on the detection of Treponema-specific IgG has
been made available. However, a nontreponemal technique
like RPR is the most widely used including the study area,
though it is unreliable as a positive result does not necessarily
indicate treponemal infection. Thus, its application in
screening blood donors, pregnant women, and social workers
has been in question. Therefore, we assessed the performance
of ECOTEST-RPR and DIALAB-ELISA assay with Randox-
TPHA as a reference standard diagnostic test for syphilis.
In the current study, the overall sensitivity and specificity
of ECOTEST-RPR as compared to Randox-TPHA were
100% and 80.8%, respectively. In our study, the sensitivity
and specificity of RPR were comparable to the findings
reported from Portugal, Korea, and Nepal [19–21]. However,
sensitivity alone was much higher compared to reports from
4 Journal of Immunology Research

Table 1: The detection of syphilis by Randox-TPHA reactivity among study participants at University of Gondar Hospital, 2015–2016.

Demography Total (%) Randox-TPHA Positive (%) Randox-TPHA Negative (%) COR (95% CI) AOR (95% CI)
Male 84 (52.5) 33 (39.3) 51 (60.7) 1.11 (0.58–2.10) 1.15 (0.59–2.25)
Gender 1
Female 76 (47.5) 28 (36.8) 48 (63.2) 1
1111111
20–30 121 (75.6) 48 (39.7) 73 (60.3) 0.76 (0.35–1.66) 0.75 (0.34–1.67)
Age (years) 31–40 36 (22.5) 12 (33.3) 24 (66.7) 1 1
≥41 3 (1.9) 1 (33.3) 2 (66.7) N/A N/N/A N/A
Urban 53 (33.1) 19 (35.8) 34 (64.2) 0.86 (0.44–1.71) 0.90 (0.44–1.83)
Residence
Rural 107 (66.9) 42 (39.3) 65 (60.7 1 1
N/A: not considered during analysis; TPHA: T. pallidum hemagglutination assay; COR: crude odds ratio; AOR: adjusted odds ratio; CI: confidence interval;
%: percent.

Table 2: Serology results of the ECOTEST-RPR and Randox-TPHA drug resistance, and social impacts in the community.
tests at the University of Gondar Hospital, 2015–2016. Besides, blood from donors that are incorrectly diagnosed
and labeled as positive will be discarded. Therefore, in the
Randox-TPHA presence of this limitation and with the availability of other
ECOTEST-RPR
Reactive Nonreactive Total
syphilis screening tests, the sole utilization of ECOTEST-
Reactive 61 19 80 RPR assay routinely as a diagnostic method remains of great
Nonreactive 0 80 80 concern in the study area.
Total 61 99 160 Nevertheless, the specificity of the test in our study
Sensitivity of ECOTEST-RPR (61 of 61 samples), 100%; specificity of showed lowest performances compared to the Indian and
ECOTEST-RPR (80 of 99 samples), 80.8%; agreement (61 + 80 = 141, of South African report (96.96% and 100%, resp.) [22, 27] and
160 samples), 88%; positive predictive value (61/80) 76.2%; and negative incomparably very high with respect to findings from Turkey
predictive value (80/80) 100%. and Latvia (0%) [23, 28]. In contrast to findings from studies,
those come up with a superior specificity of RPR [20, 27, 28],
Table 3: Serology results of the DIALAB-ELISA and Randox- and we found a low specificity of manual ECOTEST-RPR test
TPHA tests at the University of Gondar Hospital, 2015–2016. performance. The likely explanation may be the variation in
the type of methods between our study and theirs. Although
Randox-TPHA RPR is generally a nontreponemal test, we used a conven-
DIALAB-ELISA
Reactive Nonreactive Total tional manual ECOTEST-RPR test method while their
Reactive 60 5 65 method was the automated RPR test, indicating that the
Nonreactive 1 94 95 automated one has reduced interpersonal differences and
Total 61 99 160 other confounders. Furthermore, the clinical stage of study
Sensitivity of DIALAB-ELISA (60 of 61 samples), 98.4%; specificity of
participants (primary, secondary, and tertiary syphilis)
DIALAB-ELISA (94 of 99 samples), 94.9%; agreement (60 + 94 = 154, of affects the specificity of the test because of the prozone phe-
160 samples), 96.25%; positive predictive value (60/65) 92.3%; and negative nomenon. Moreover, since reporting of the RPR test result
predictive value (94/95) 98.9%. (as positive or negative) is based on observation, the subjec-
tive (interpersonal) variation and subsequent decisions
several studies [22–26]. The highest (100%) sensitivity in our between laboratory analysts potentially affect the specificity
finding could be as follows: First, our participants may not of the test result.
have only syphilis infection, suggesting cross-reactivity of Following the innovative introduction of ELISA and
ECOTEST-RPR with cholesterol, lecithin, and cardiolipin recombinant DNA (rDNA) technology for the diagnosis of
antigens found in other disease processes as a result of cellu- syphilis, the traditional two-step approach of first screening
lar destruction. Second, it may be due to variation in proto- with a nontreponemal test and then using a Treponema-spe-
cols from different companies as well; in fact, it should not cific confirmation test has been confronted and, as result, two
be forgotten that the diagnostic performance of RPR is pro- diagnostic thoughts appeared. One school promotes the use
foundly affected by the stages of syphilis infection, which is of EIA as a screening test which needed to be confirmed by
not fully addressed in our study. Based on our finding, the another test (technology or method but with equal or higher
highest sensitivity (100%) is considered as a limitation of specificity), and the other school proposes the traditional
the ECOTEST-RPR serological test as its false positivity is approach of an algorithm [29].
enormous due to cross-reactive antibodies. Also, the chance Accepting either the traditional or the other approach,
of missing syphilitic cases at different stages is there because evaluating the performance of ELISA with reference to a bet-
of the prozone effect. This high sensitivity of RPR has a ter method is compulsory. In contrast to the ECOTEST-RPR
negative implication in that wrongly diagnosed individuals test, DIALAB-ELISA has come up with a better test perfor-
are supposed to take medication together with their sexual mance with reference to Randox-TPHA. In this study, we
partners (if there); this phenomenon results in economical, found 5 false positives with the DIALAB-ELISA test, thus
Journal of Immunology Research 5

giving a false-positive diagnostic rate of 8%; this appears to be enlighten the readers not to underestimate the drawbacks
the least value as compared to that of RPR (31%). of ELISA methods.
The sensitivity and specificity of DIALAB-ELISA with
respect to Randox-TPHA are 98.4% and 94.9%, respectively. 5. Conclusion
Findings obtained from several studies have come up with a
sensitivity and specificity ranging from 90 to 100%, by far a This study comes up with a characteristically variable diag-
result that is aligned with our study [23, 28–32]. Despite these nostic performance of the DIALAB-ELISA test and the cur-
similarities, it should not be forgotten that other findings rently available traditional ECOTEST-RPR test in Ethiopia
[33–35] on the performance of ELISA moderately differ from as compared to Randox-TPHA. ELISA kits with the recombi-
the results of the current study. The most probable reason for nant T. pallidum antigens have certain attractions as a diag-
this performance variation may be due to the type of immu- nostic tool. However, we cautioned the efficacy of the
nodominant syphilis proteins that are incorporated into the independent application of both ECOTEST-RPR and ELISA
wells of ELISA kits. as a screening/diagnostic test for syphilis infection. In addi-
During the evaluation of comparative prediction of a tion, it is important to underline that healthcare providers
diagnostic kit with a reference standard, several issues will must perform a thorough review of each patient’s clinical
influence the interpretation of their results of which the and treatment history while choosing the type of test and
prevalence/magnitude of a disease is the most important interpreting the results of RPR and ELISA IgG/IgM tests
factor [36]. for syphilis diagnosis. Consequently, thorough studies should
In the current study, the positive and negative predictive be conducted, aiming for a change of the current diagnostic
values of ECOTEST-RPR are 76.2 and 100%, respectively. In scheme used in the community.
the presence of variability on study participants (considering
50% of participants were positive for ECOTEST-RPR in this Abbreviations
study), several studies come up with predictive values of
ECOTEST-RPR similar with those of the current study [19, CIs: Confidence intervals
21, 26] whereas different prediction values of ECOTEST- EIA: Enzyme immunoassay
RPR have been reported from other studies [23]. The PPV ELISA: Enzyme-linked immunosorbent assay
and NPV of DIALAB-ELISA were 92.3% and 98.9%, respec- FTA-abs: Fluorescent treponemal antibody absorption test
tively, in this study. Likewise, reports from studies showed IgG: Immunoglobulin G
PPVs and NPVs which are comparable with ours [23, 29, IRB: Institutional Review Board
34], whereas variable predictive values were seen from a NPV: Negative predictive value
Turkish study [23]. As we have stated earlier, the predictive NAAT: Nucleic acid amplification techniques
values in this study (for ECOTEST-RPR and DIALAB- PCR: Polymerase chain reaction
ELISA) were comparable and variable while comparing with PPV: Positive predictive value
PVs from several studies; a crucial point is that prevalence RPR: Rapid plasma reagin
affects the PVs of any test. This implies that the same diag- rDNA: Recombinant DNA
nostic test will have a different predictive accuracy according STARD: Standards for Reporting Diagnostic Accuracy
to the clinical setting/nature of study participants; thus, we Studies
strongly enlighten that the recommendations made in this STD: Sexually transmitted disease
study are based on the 50% magnitude of syphilis and that TPHA: T. pallidum hemagglutination
other studies should recognize the influence of the prevalence T pallidum: Treponema pallidum
of disease when considering predictive values of diagnostic or UGH: University of Gondar Hospital
screening tests. VDRL: Venereal disease research laboratory.
The reported test performance of DIALAB-ELISA
(sensitivity, specificity, and predictive values) in this study Data Availability
is encouraging and makes the test a better choice of the
diagnosis approach in syphilis-suspected cases. Moreover, The authors confirm that all data supporting our findings
the agreement between DIALAB-ELISA and TPHA was are contained within the manuscript and are fully available
nearly perfect (kappa value 0.96) as compared to that of without restriction.
ECOTEST-RPR and Randox-TPHA (kappa value of 0.88).
Besides the superior performance, the ELISA technique has Ethical Approval
plenty of advantages over conventional flocculation screening
tests (RPR). The method is designed potentially to be auto- Ethical clearance was obtained from the University of
mated plus the reading of results is usually carried out by Gondar Research Ethics Committee. Participation was vol-
a microtiter plate reader, thus making the interpretation untary, and informed consent was obtained from all subjects
of results objective, unlike that of the RPR test which is sub- who accepted to participate in the study. Participants’ will-
jective and hence requires having extensive experience. ingness was asked verbally after briefly explaining the objec-
Unlike RPR, concerns like the prozone phenomenon and tives of the study, the risks and benefits of the procedures,
stage of syphilis infection do not affect the ELISA method. and the right not to participate in the study using their local
Regardless of all the above rational benefits, we want to language. The authors received verbally informed consent
6 Journal of Immunology Research
before including any of the participants in this study. Written
consent was not acquired because syphilis-positive partici-
pants were recruited from the outpatient department labora-
tory of the Gondar University Hospital where they were sent
to undergo a syphilis antibody test. Similarly, 80 apparently
healthy participants with no history of sexually transmitted
diseases were recruited from the hospital blood bank; thus,
in both groups, we did not took any additional specimen
but rather used the already provided blood sample that they
provide at the hospital laboratory and blood bank. The addi-
tional sociodemographic data collection was a noninvasive
procedure with no risk associated to it. Therefore, consider-
ing all these facts, only verbal agreement was acquired to be
included in the study. Thereafter, only voluntary participants
who are willing to give us a verbal consent (agree to partici-
pate) were recruited into the study.
Conflicts of Interest
The authors declare that they have no competing interest
with regard to the present study.
Authors’ Contributions
Markos Negash and Demeke Geremew conceived the study
concept and designed the study; Markos Negash and Tadelo
Wondmagegn carried out the data collection and laboratory
analysis; Demeke Geremew and Tadelo Wondmagegn super-
vised the data collection and laboratory analysis; Markos
Negash, Demeke Geremew, and Tadelo Wondmagegn ana-
lyzed the data and prepared the first manuscript draft; and
Markos Negash and Demeke Geremew reviewed the draft.
All authors read and approved the final manuscript.
Acknowledgments
The authors would like to acknowledge their technical
assistant, Mister Amare Kifle, for his excellent technical
support during the conduct of the study. The authors’ grat-
itude also goes to all participants in the study and the Uni-
versity of Gondar Hospital. The authors are also thankful
to the Gondar Blood Bank Center staff for their unreserved
support during the study.
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